PROJECT SUMMARY Pediatric tumors represent a very heavy social and economic burden with profound emotional involvement not only for the directly affected children but also for their family and friends. Within pediatric tumors, neuroblastoma (NB) is of particular relevance, since it is the most common solid cancer in children outside of the skull and it still kills about 40% of patients diagnosed with the most aggressive forms. Therefore, there is a clear need to improve the treatment for this deadly disease. One of the strategies to achieve this goal is to overcome the emergence of resistance to chemotherapy by cancer cells. Published data show that Tumor-Associated Macrophages (TAMs) provide positive support to NB cells and increase their resistance to chemotherapy. The mechanisms that mediate this support and the communication vehicles between these cell types are poorly understood. My preliminary data have started to elucidate this, through the following findings: 1) NB cells secrete exosomes that contain microRNAs (miRs); 2) The miRs are taken up by surrounding macrophages and can bind to Toll-like receptor 8 (TLR8), triggering TLR8 activation in macrophages; 3) TLR8 stimulation causes macrophages to secrete exosomes that contain specific miRs; 4) the NB cells uptake TAM-derived exosomic miRs which silence BRG1, RHOB, and FBXW7 in NB cells. These proteins are well known inhibitors of MYC (aka as c-MYC) and MYCN expression in NB; 5) the levels of MYC and MYCN are significantly higher in NB cells co-cultured with human monocytes and in primary NBs with higher TAM infiltration. NB is a very MYC-driven tumor. It is known that MYCN increases multi-drug resistance in NB. We provide evidence that also MYC promotes NB resistance to chemotherapy both in vitro and in vivo. We also identified a panel of cytokines secreted by TAMs when co-cultured with NB cells, which could contribute to the up-regulation of MYC and MYCN in NB cells when co-cultured with human monocytes. Therefore, our hypothesis is that TAMs secrete soluble factors (exosomic miRs and cytokines) that induce up- regulation of MYC and MYCN in NB cells, increasing their resistance to chemotherapy. This proposal has three Specific Aims. In the first we will examine the mechanisms by which exosomic miRs induce resistance to therapy in NB. Then we will assess the therapeutic potential of targeting TAM-derived soluble factors (exosomic miRs and cytokines) to overcome NB resistance. Finally, we will correlate circulating TAM-derived exosomes in the blood of NB patients with the degree of TAM infiltration in the primary tumor, traditional clinical imaging techniques and clinical outcome. I have brought together a team of seasoned scientists with well- established expertise in NB and in the field of miRs and cancer therapeutics. This research will lead to the identification of new molecular targets, the development of a new strategy to overcome drug resistance in NB (and possibly other types of MYC-driven cancers), and the possible definition of new circulating biomarkers to identify subsets of NB patients suitable for an anti-TAM therapy.